No abstract

Monoterpene indole alkaloids (MIAs) are an expansive class of plant natural products, many of which have been named on the World Health Organization's List of Essential Medicines. Low production from native plant hosts necessitates a more reliable source of these drugs to meet global demand. Here, we report the development of a yeast-based platform for high-titer production of the universal MIA precursor, strictosidine. Our fedbatch platform produces ∼50 mg/L strictosidine, starting from the commodity chemicals geraniol and tryptamine. The microbially produced strictosidine was purified to homogeneity and characterized by NMR. Additionally, our approach enables the production of halogenated strictosidine analogues through the feeding of modified tryptamines. The MIA platform strain enables rapid access to strictosidine for reconstitution and production of downstream MIA natural products.

We report biosynthetic pathways that can synthesize and transform conjugated octaenes and nonaenes to complex natural products. The biosynthesis of (−)-PF1018 involves an enzyme PfB that can control the regio-, stereo-, and periselectivity of multiple reactions starting from a conjugated octaene. Using PfB as a lead, we discovered a homologous enzyme, BruB, that facilitates diene isomerization, tandem 8π-6π-electrocyclization, and a 1,2-divinylcyclobutane Cope rearrangement to generate a new-to-nature compound.

Genome mining of biosynthetic pathways with no identifiable core enzymes can lead to discovery of the so-called unknown (biosynthetic route)-unknown (molecular structure) natural products. In this work, we focused on a conserved fungal biosynthetic pathway (ank) that lacks a canonical core enzyme, and used heterologous expression to identify the associate natural product to be a highly modified cyclo-arginine-tyrosine dipeptide (cRY). Biochemical characterization of the ank pathway led to identification of a new arginine-containing cyclodipeptide synthase (RCDPS), which was previously annotated as a hypothetical protein (HP) and has no sequence homology to nonribosomal peptide synthetase (NPRS) or bacterial cyclodipeptide synthase (CDPS). RCDPS homologs are widely encoded in fungal genomes and we showed other members of this family can synthesize diverse cyclo-arginine-Xaa dipeptides. Characterization of a cyclo-Arg-Trp (cRW) RCDPS showed the enzyme is aminoacyl-tRNA dependent, and represents the first report of such CDPS-like enzyme from fungi. Further characterization of the biosynthetic pathway anchored by the cRW synthase led to discovery of new compounds of which the structures would not have been predicted without knowledge of RCDPS function.

The structural determination of natural products (NPs) can be arduous due to sample heterogeneity. This often demands itera-tive purification processes and characterization of complex molecules that may only be available in miniscule quantities. Microcrystal electron diffraction (microED) has recently shown promise as a method to solve crystal structures of NPs from nanogram quantities of analyte. However, its implementation in NP discovery remains hampered by sample throughput and purity requirements akin to traditional NP-discovery workflows. In the methods described herein, we leverage the resolving power of transmission electron microscopy (TEM) and the miniaturization capabilities of DNA microarray technology to address these challenges through the establishment of an NP screening platform, array electron diffraction (ArrayED). In this workflow, an array of HPLC fractions taken from crude extracts are deposited onto TEM grids in picoliter-sized droplets. This multiplexing of analytes on TEM grids enables 1200 or more unique samples to be simultaneously inserted into a TEM equipped with an autoloader. Selected area electron diffraction analysis of these microarrayed grids allows for rapid identification of crystalline metabolites. In this study, ArrayED enabled structural characterization of 14 natural products, including four novel crystal structures and two novel polymorphs, from 20 crude extracts. Moreover, we identify several chemical species that would not be detected by standard mass spectrometry (MS) or UV/Vis and crystal forms that would not be characterized using traditional methods.